Given that it is necessary to amplify a DNA fragment of the sex chromosome, to obtain enough sensitivity in order to determine the embryo's sex from a few sampled cells, it is necessary to discuss the molecular mechanisms of DNA amplification.
Any simple DNA chain has two terminals (or extremes), one of them with phosphate in carbon 5' of the deoxyribose molecule (extreme 5'), the other with hydroxyl in position 3' of the sugar (extreme 3'). At the same time, the complementary chain has the same kind of extremes in opposing positions, that is, the extreme 3' of a chain has the other 5' in front of it. For this reason, it is considered that the two complementary chains of a DNA molecule have opposite polarities and are antiparallel.
It is known that the replication of a DNA chain is produced by the combination of a nucleotide-5'-triphosphate with the extreme 3' of the chain. Therefore, this extreme 3' is the growing point of the DNA, and synthesis proceds in opposite directions in both complementary chains.
A template chain is simply a DNA chain. However, the template chain doesn't provide any extreme 3' able to promote the beginning of the growth of the new chain that is going to be synthesized. For this reason, the synthesis of a new DNA molecule is only possible in the presence of a nucleic acid fragment that can provide the extreme 3'. The name given to this nucleic acid fragment is "primer."
The DNA synthesis mechanisms discussed above require the action of a series of enzymes that catalyze the reaction. The existence of polymerases has been proven among the enzymes that have been identified.
The application to lab techniques of what has been discussed was perfected in the so-called PCR (Polymerase Chain Reaction) [3]. In each step of this technique, the number of chains obtained in the former step, is doubled, so this number has a geometric growth.
However, the application of this technique has one limitation derived from the synthesis, of spurious DNA fragments from the primers themselves which don't correspond to the template that one wants to copy. This can be attributed to contamination, as well as by other causes. But the correct copies of the template, as well as the false ones, have in their terminals the sequences of the primers themselves, which create complementary sequences that, in the next step, will be amplified. Therefore, in the following steps the target chains that are under consideration, as well as the false ones, amplify, and there comes a point where it is impossible to improve the sensitivity.
In the present invention's procedure, the polymerase chain reaction PCR technique is improved as follows.
After the first round of the polymerase chain reaction PCR technique on a DNA fragment, there comes a point in which it is impossible to improve the sensitivity of the technique. Experimentally, it was found that approximately 45 cycles is the upper limit of amplification.
To improve the relation between the number of similar chains of the original target and the number of false chains, it is necessary to distinguish the first ones, as the second ones won't necessarily be homogeneous. According to the present invention it is possible to do it looking for a smaller DNA fragment, whose sequence is a subsequece of the original sequence. For that purpose, other DNA chains, whose sequences correspond to sequences of the chains under consideration, are used as new primers. In this way, the polymerase chain reaction PCR technique is applied again, with its geometric growth, but only on the DNA amplified fragments that correspond to the original template.